The overall objective of this proposed research is to establish the mechanisms responsible for the regulation of leucine oxidation by mammalian cells. Studies will be conducted with isolated hepatocytes, perfused rat heart, isolated liver and heart mitochondria, and branched-chain alpha-keto acid dehydrogenase complex isolated from liver or heart tissue. The specific aims include to establish: (a) whether the branched-chain alpha-keto acid dehydrogenase complex is subject to conversion between active and inactive forms by a phosphorylation-dephosyphorylation cycle; (b) an assay for the branched-chain alpha-keto acid dehydrogenase complex which will measure the total activity of the enzyme in liver, heart, muscle, and adipose tissue; (c) an assay for the branched-chain alpha-keto acid dehydrogenase complex which will measure the percentage of the enzyme in the active form in these tissues; (d) whether fasting, obesity, diabetes, or protein deprivation change the percentage of the branched-chain alpha-keto acid dehydrogenase complex in the active form in these tissues; (e) the relationship between the regulation of the pyruvate dehydrogenase complex and the regulation of the branched-chain alpha-keto acid dehydrogenase complex; (f) whether dichloroacetate and 2-chloropropionate are inhibitors of the branched-chain alpha-keto acid dehydrogenase kinase; (g) whether the various branched-chain keto acids are inhibitors of the branched-chain alpha-keto acid dehydrogenase kinase; and (h) the mechanism responsible for the apparent inhibition by dichloracetate of branched-chain amino acid catabolism by skeletal muscle. The major working hypothesis of the study is that the branched-chain alpha-keto acid dehydrogenase complex is subject to regulation by a phosphorylation cycle.